CN109438572A - The method of continuous application affinity chromatography and ceramic hydroxyapatite chromatography antibody drug - Google Patents
The method of continuous application affinity chromatography and ceramic hydroxyapatite chromatography antibody drug Download PDFInfo
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- CN109438572A CN109438572A CN201811194791.0A CN201811194791A CN109438572A CN 109438572 A CN109438572 A CN 109438572A CN 201811194791 A CN201811194791 A CN 201811194791A CN 109438572 A CN109438572 A CN 109438572A
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- buffer
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
Abstract
The method that the present invention discloses a kind of continuous application affinity chromatography and ceramic hydroxyapatite chromatography antibody drug, comprising steps of 1) sodium phosphate buffer salt is added in by the antibody-solutions after affinitive layer purification to final concentration of 5-15mM, it is 6.7~6.9 with the pH that Tris-base adjusts antibody-solutions, is 5~7mS/cm with the conductivity that NaCl adjusts antibody-solutions；2) antibody-solutions for obtaining step 1) are chromatographed using ceramic hydroxyapatite；The step of ceramic hydroxyapatite chromatographs are as follows: a, handle affine filler, loading；B, it is chromatographed using elution 2 buffers elution column；Elute the Buffer-B that 2 buffers are volume fraction 10%；C, it elutes, first uses Buffer-B gradient elution, reuse 100%Buffer-B elution.Affinity chromatography can be used in conjunction in method of the invention, carry out two-step purifying, simplify the purifying process of albumen, reduce costs, and greatly shorten the time；Also have the characteristics that high resolution, the purity of albumen can be increased to 90% or more.
Description
Technical field
The present invention relates to field of biological pharmacy, are not also to be related to a kind of method of the separation and purification of antibody drug.
Background technique
In antibody drug R&D process, the isolation and purification of antibody is committed step.In antibody preparation process, first
Carry out rich protein, such as Protein A and Protein G affinity purification.During this, low pH value is needed to be eluted.
At low PH, protein is easy to produce poly, especially for bispecific antibody.Therefore, seek the quickly and effectively side of separation
Formula has very important significance for antibody producing with the polymer reduced in antibody.
Hydroxyapatite, is a kind of calcium phosphate inorganic compound that nature is widely present, and molecular formula is (Ca5(PO4)3
(OH))2.Natural hydroxyapatite can be used as protein chromatographic column with protein, the interaction such as polypeptide and nucleic acid
Filled media.But native hydroxyl apatite stability is poor, service life is shorter.Kadoya in 1986 etc. is by calcination method
It introduces, develops ceramic hydroxyapatite, and use initially as chromatographic column medium.
Although ceramic hydroxyapatite has some of the above advantage, it is not a kind of succinct chromatography to a certain extent
Packing material, compared to single mode interaction chromatographic material for, the research and development in relation to it are relatively difficult.It makes pottery accordingly, with respect to CHT
Porcelain hydroxyapatite column is used for separation antibody drug in conjunction with affinity chromatography, and there is no applied by most of researcher.
Summary of the invention
Technical problem to be solved by the present invention lies in, a kind of method of separation antibody drug is provided, it can be fast and effective
Removal antibody in polymer, obtain the antibody drug that polymer is substantially reduced, and greatly shorten purification time.
In order to solve the above technical problems, the present invention provides a kind of continuous application affinity chromatography and ceramic hydroxyapatite chromatography
The method of separation antibody drug, the described method comprises the following steps:
1) sodium phosphate buffer salt is added in by the antibody-solutions after affinitive layer purification to final concentration of 5-15mM,
It is 6.7~6.9 with the pH that Tris-base adjusts antibody-solutions, is 5~7mS/cm with the conductivity that NaCl adjusts antibody-solutions；
2) antibody-solutions for obtaining step 1) are chromatographed using ceramic hydroxyapatite.The ceramic hydroxyapatite chromatography
The step of are as follows:
A, affine filler, loading are handled；
B, it elutes, is chromatographed using elution 2 buffers elution column；2 buffers of the elution are volume fraction 10%Buffer-
B+90%Buffer-A；The Buffer-B is the solution of 10mM sodium phosphate buffer salt, 1M sodium chloride, pH 6.8；It is described
Buffer-A is the solution of 10mM sodium phosphate buffer salt, 30mM sodium chloride, pH 6.8；
C, it elutes, first carries out gradient elution, Buffer-B volume fraction using the dicyandiamide solution of Buffer-B+Buffer-A
90%, Buffer-A volume fraction is increased to by 10% gradient and is reduced to 10% by 90% gradient；Reuse 100%Buffer-B
Product is collected in elution.
In an embodiment of the present invention, in step 1), the affinitive layer purification include protein A affinity chromatography and
Protein G is affine；Preferably, the affinitive layer purification refer to antibody drug by protein A affinity chromatography and
Protein G affinity chromatography is purified.
In an embodiment of the present invention, in step 1), the final concentration of 8-12mM of sodium phosphate buffer salt；Preferably, phosphoric acid
The final concentration of 10mM of sodium buffer salt.
In an embodiment of the present invention, in step 1), the pH to 6.8 of antibody-solutions is adjusted with Tris-base.
In an embodiment of the present invention, in step 1), the conductivity of antibody-solutions is adjusted to 6mS/cm with NaCl.
In an embodiment of the present invention, in step 2), ceramic hydroxyapatite chromatography uses spherical ceramic hydroxyapatite
(CHT) filler, the filler have two basic change site, including five calcium doublets (site C) and a pair of phosphorus three containing hydroxyl
Conjuncted (site P).
In an embodiment of the present invention, in step a, handling affine filler includes three elution, pretreatment and balance operations.
Wherein, elution is using ddH2O solution elutes column chromatography；Pretreatment is the sodium phosphate buffer salting liquid using pH 6.8,500mM
Handle column chromatography；Balance is using Buffer-A, and the Buffer-A is 10mM sodium phosphate buffer salt, 30mM sodium chloride, pH
6.8 solution.Wherein, Buffer-A dosage is 5-15 times of column volume, preferably 10 times of column volumes.
In an embodiment of the present invention, in step b, 2 buffer dosages of elution are 2-10 times of column volume, preferably 5 times of columns
Volume.
In an embodiment of the present invention, in step c, solvent gradient dosage is 10-30 column volume, preferably 20 cylinders
Product.
In an embodiment of the present invention, step d is additionally provided with after step c: chromatographic column being balanced and is saved.Specifically
, in the step d, the residual protein in column chromatography first is cleaned with Buffer C, then store chromatographic column with Buffer D, it is described
Buffer C is pH 6.8,500mM sodium phosphate buffer, and the Buffer D is 500mM NaOH solution.Wherein, Buffer C
Dosage is 2-10 times of column volume, preferably 5 times of column volumes；Buffer D dosage is 1-3 times of column volume, preferably 2 times of column volumes.
In an embodiment of the present invention, step 2) need to carry out under 20~30 degrees Celsius, cannot make in 4 DEG C of chromatography cabinets
With.
In an embodiment of the present invention, except loading and elution speed are 100cm/hr, other process flow velocitys are up to 300cm/
hr。
In embodiments of the present invention, antibody is after protein A and protein G affinity purification, albumen institute
It is that directly solution can be adjusted in low pH Glycine in environment, carries out two step purifying to reach and CHT is directly used in conjunction
Effect.Then antibody-solutions are adjusted with 500mM buffer solution of sodium phosphate (NaPhosphate), make in albumen containing eventually
Concentration is the buffer solution of sodium phosphate (NaPhosphate) of 10mM.Because of the particularity of CHT medium, buffer needs certain
The NaPhosphate of concentration, and the experiment condition of 5-10mM NaPhosphate is for the stability and height of CHT chromatographic column
Resolution ratio is necessary.Lead protein salt for 6mS/cm with 2M Tris-base titration PH to 6.8,5M NaCl titration.
The use of ceramic hydroxyapatite chromatography is a kind of spherical ceramic hydroxyapatite (CHT) filler in the present invention, it includes
Two basic change site, including five calcium doublets (site C) and a pair of phosphorus triplet (site P) containing hydroxyl.The ceramics material
Material overcomes limitation of the conventional crystal material hydroxyapatite in large-scale industrial production, imitates with unique separation
Fruit, while can be reused under high flow rate, large-scale chromatographic column up to hundreds of times.
Continuous application affinity chromatography provided by the invention and ceramic hydroxyapatite chromatograph Ceramic
The method of Hydroxyapatite (CHT) separation antibody drug, has the advantage that
(1) for the high carrying capacity of protein, still have under high flow rate highly selective.
(2) affinity chromatography can be used in conjunction, carrying out two-step purifying to simplify protein purification process reduces cost, greatly
Shorten the time.
(3) purity of albumen is increased to 90% or more by high resolution, and can be adsorbed affinity chromatography and be brought into the process
Protein A, nucleic acid and endotoxin etc..
(4) rate of recovery is high, and in more than ten of the project purified, the sample average rate of recovery can reach 70% or so.
(5) it can amplify for downstream process scale and useful data is provided, improve the consistency and stability of experiment.
Detailed description of the invention
Fig. 1 is that the CHT of embodiment 3 purifies instance graph one.
Fig. 2 is that the CHT of embodiment 3 purifies instance graph two.
Specific embodiment
Clear, complete description will be carried out to technical solution of the present invention below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
The selection of embodiment 1CHT chromatographic column.
CHT chromatographic column is divided into two major classes according to type, can choose corresponding chromatographic column according to demand.CHT ceramics hydroxyl phosphorus
Lime stone medium (I type and II type) medium has 20,40 and 80um, tri- kinds of sizes.The characteristic of two kinds of CHT medium is similar, but
Also there is the difference of part.CHT I type carrying capacity is high, and it is stronger for acidic protein binding force, can be used in removal virus,
DNA, endotoxin and polymer.II type carrying capacity wants low with respect to I type, but its resolution ratio is higher, anti-suitable for more hypotypes are purified
Body.When protein multimers proportion is bigger, the carrying capacity of albumen can be accordingly reduced, to need to reduce applied sample amount, to reach
To higher resolution ratio.
Embodiment 2 adjusts sample, realizes that two step chromatographies are used in conjunction.
For antibody after protein A and protein G affinity purification, environment where albumen is low pH Glycine
In.Directly solution can be adjusted, to achieve the effect that CHT, which is directly used in conjunction, carries out two step purifying.Use 500mM
NaPhosphate is adjusted, and albumen is made to contain 10mM NaPhosphate.Because of the particularity of CHT medium, buffer is needed
Certain density NaPhosphate is wanted, and the experiment condition of 5-10mM NaPhosphate is for the stability of CHT chromatographic column
And high-resolution is necessary.Lead protein salt for 6mS/cm with 2M Tris-base titration PH to 6.8,5M NaCl titration.
These parameters can effectively be very important protein sample in conjunction with chromatographic column.
Embodiment 3CHT purifies example.
The pure processing step of CHT is as follows:
Wherein, buffer A balances 10 times of column volumes of chromatographic column, and 10% buffer B carries out the clear of 5 times of column volumes
It washes, the buffer B linear gradient elution of 20 times of column volumes, the buffer C of 5 times of column volumes cleans residual protein, 2 times of column volumes
Buffer D store chromatographic column.It is as shown in Figs. 1-2 that CHT purifies instance graph.Shown in Fig. 1-2, Y-axis is 280nm (A280)
Absorption value；X-axis is that first peak 1 liquid volume (mL) is antibody monomer, and second peak 2 is polymer, and third peak 3 is more
Aggressiveness and impure impurity.The purity of albumen is increased to 90% or more after purified, and sample recovery rate can reach 70% or more.
In whole process of purification, it cannot be used in 4 DEG C of chromatography cabinets, in order to avoid 500mM NaPhosphate crystalline damage
Chromatographic column need to carry out at room temperature.Except loading and elution speed are 100cm/hr, other process flow velocitys are up to 300cm/hr.
In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, it is not of the invention to limit
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in
In protection scope of the present invention.
Claims (15)
1. a kind of method of continuous application affinity chromatography and ceramic hydroxyapatite chromatography antibody drug, which is characterized in that
The following steps are included:
1) sodium phosphate buffer salt is added in by the antibody-solutions after affinitive layer purification to final concentration of 5-15mM, uses
The pH that Tris-base adjusts antibody-solutions is 6.7~6.9, is 5~7mS/cm with the conductivity that NaCl adjusts antibody-solutions；
2) antibody-solutions for obtaining step 1) are chromatographed using ceramic hydroxyapatite；The step of the ceramic hydroxyapatite chromatography
Suddenly are as follows:
A, affine filler, loading are handled；
B, it elutes, is chromatographed using elution 2 buffers elution column；2 buffers of the elution are the Buffer-B+ of volume fraction 10%
90%Buffer-A；The Buffer-B is the solution of 10mM sodium phosphate buffer salt, 1M sodium chloride, pH 6.8；The Buffer-
A is the solution of 10mM sodium phosphate buffer salt, 30mM sodium chloride, pH 6.8；
C, elute, first using Buffer-B+Buffer-A dicyandiamide solution carry out gradient elution, Buffer-B volume fraction by
10% gradient is increased to 90%, Buffer-A volume fraction and is reduced to 10% by 90% gradient；100%Buffer-B is reused to wash
It is de-, collect product.
2. the method as described in claim 1, which is characterized in that in step 1), the affinitive layer purification includes protein A
Affinity chromatography and protein G are affine.
3. method according to claim 2, which is characterized in that the affinitive layer purification, which refers to, passes through antibody drug
Protein A affinity chromatography and protein G affinity chromatography are purified.
4. the method as described in claim 1, which is characterized in that in step 1), the final concentration of 8- of sodium phosphate buffer salt
12mM；Preferably, the final concentration of 10mM of sodium phosphate buffer salt.
5. the method as described in claim 1, which is characterized in that in step 1), with Tris-base adjust antibody-solutions pH to
6.8。
6. the method as described in claim 1, which is characterized in that in step 1), with NaCl adjust antibody-solutions conductivity to
6mS/cm。
7. as the method according to claim 1 to 6, which is characterized in that in step 2), ceramic hydroxyapatite chromatography makes
With spherical ceramic hydroxyapatite filler, the filler has two basic change site, and one of binding site is five calcium duplexs
Body, another binding site are a pair of phosphorus triplet containing hydroxyl.
8. the method for claim 7, which is characterized in that in step a, handle affine filler include elution, pretreatment and
Balance three operations；Elution is chromatographed using ddH2O solution elution column；Pretreatment is slow using the sodium phosphate of pH 6.8,500mM
Rush salting liquid processing column chromatography；Balance is using Buffer-A, and the Buffer-A is 10mM sodium phosphate buffer salt, 30mM chlorination
The solution of sodium, pH 6.8.
9. method according to claim 8, which is characterized in that Buffer-A dosage is 5-15 times of column volume；Preferably,
Buffer-A dosage is 10 times of column volumes.
10. the method for claim 7, which is characterized in that in step b, 2 buffer dosages of elution are 2-10 times of cylinder
Product；Preferably, 2 buffer dosages of elution are 5 times of column volumes.
11. the method for claim 7, which is characterized in that in step c, solvent gradient dosage is 10-30 times of cylinder
Product；Preferably, solvent gradient dosage is 20 times of column volumes.
12. the method for claim 7, which is characterized in that be additionally provided with step d after step c: being balanced to chromatographic column
And preservation.
13. method as claimed in claim 12, which is characterized in that in the step d, first cleaned in column chromatography with Buffer C
Residual protein, then with Buffer D store chromatographic column, the Buffer C be pH 6.8,500mM sodium phosphate buffer, it is described
Buffer D is 500mM NaOH solution.
14. method as claimed in claim 13, which is characterized in that Buffer C dosage be 2-10 times of column volume, preferably 5 times
Column volume；Buffer D dosage is 1-3 times of column volume, preferably 2 times of column volumes.
15. the method for claim 7, which is characterized in that step 2) carries out under 20~30 degrees Celsius.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110066314A (en) * | 2019-04-01 | 2019-07-30 | 上海药明生物技术有限公司 | A kind of efficient affinity purification technique for improving polymer separation resolution ratio |
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| CN105792844A (en) * | 2013-10-08 | 2016-07-20 | 第三共株式会社 | Combination of anti-FGFR2 antibody and other agent |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066314A (en) * | 2019-04-01 | 2019-07-30 | 上海药明生物技术有限公司 | A kind of efficient affinity purification technique for improving polymer separation resolution ratio |
| CN110066314B (en) * | 2019-04-01 | 2023-10-27 | 上海药明生物技术有限公司 | Affinity purification process for efficiently improving separation resolution of multimer |
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Application publication date: 20190308 |